Identification of Microorganisms

• For many students and professionals the most

pressing topic in microbiology is how to identify
unknown specimens.
• Why is this important?
• Labs can grow, isolate and identify most routinely
encountered bacteria within 48 hrs of sampling.
• The methods microbiologist use fall into three
categories:
♣ Phenotypic- morphology (micro and
macroscopic)
♣ Immunological- serological analysis
♣ Genotypic- genetic techniques
1
 Microbe Identification
• Identification measures include:
♣ Microscopy (staining)
♣ growth on enrichment, selective, differential or
characteristic media
♣ specimen biochemical test (rapid test methods)
♣ immunological techniques
♣ molecular (genotypic) methods.
• After the microbe is identified for clinical
samples it is used in susceptibility tests to
find which method of control is most
effective.
2
 Phenotypic Methods
• ‘Old fashioned’ methods via biochemical,
serological and morphological are still used
to identify many microorganisms.
• Phenotypic Methods
• Microscopic Morphology include a
combination of cell shape, size, Gram stain,
acid fast rxn, special structures e.g.
endospores, granule and capsule can be
used to give an initial putative
identification.
3
 Phenotypic Methods
• Macroscopic morphology are traits that can be
accessed with the naked eye e.g. appearance of
colony including texture, shape, pigment, speed of
growth and growth pattern in broth.
• Physiology/Biochemical characteristic are
traditional mainstay of bacterial identification.
• These include enzymes (catalase, oxidase,
decarboxylase), fermentation of sugars, capacity
to digest or metabolize complex polymers and
sensitivity to drugs can be used in identification.
4
 Genotypic Methods
• Genotypic methods involve examining the
genetic material of the organisms and has
revolutionized bacterial identification and
classification.
• Genotypic methods include PCR (RT-PCR,
RAPD-PCR),use of nucleic acid probes, RFLP
and plasmid fingerprinting.
• Increasingly genotypic techniques are
becoming the sole means of identifying
many microorganisms because of its speed
and accuracy. 5
 Microbe
Identification

6
Phenotypic Methods of Identification
• Microbiologists use 5 basic techniques to grow,
examine and characterize microorganisms in
the lab.
• They are called the 5 ‘I’s: inoculation,
incubation, isolation, inspection and
identification.
• Inoculation: to culture microorganisms a tiny
sample (inoculum) is introduced into medium
(inoculation).
• Isolation involves the separating one species
from another.
7
Phenotypic methods of Identification

• Incubation: once the media is inoculated it

is incubated which means putting the culture
in a controlled environment (incubation) to
allow for multiplication.
• After incubation the organisms are
inspected and identified phenotypically,
immunologically or genetically.

8
 Genotypic methods
• Genotypic methods of microbe
identification include the use of :
Nucleic acid probes
PCR (RT-PCR, RAPD-PCR)
Nucleic acid sequence analysis
rRNA analysis
RFLP
Plasmid fingerprinting.

9
 Nucleic acid probes
• Nucleic acid hybridization is one of the most
powerful tools available for microbe
identification.
• Hybridization detects for a specific DNA
sequence associated with an organism.
• The process uses a nucleic acid probe which
is specific for that particular organism.
• The target DNA (from the organism) is
attached to a solid matrix such as a nylon or
nitrocellulose membrane. 10